Analogs of Gastric Inhibitory Polypeptide and Their Use for Treatment of Diabetes

ABSTRACT

The present invention provides peptides which stimulate the release of insulin. The peptides, based on GIP 1-42 include substitutions and/or modifications which enhance and influence secretion and/or have enhanced resistance to degradation. The invention also provides a process of N terminally modifying GIP and the use of the peptide analogues for treatment of diabetes.

The present invention relates to the release of insulin and the controlof blood glucose concentration. More particularly the invention relatesto the use of peptides to stimulate release of insulin, lowering ofblood glucose and pharmaceutical preparations for treatment of type 2diabetes.

Gastric inhibitory polypeptide (GIP) and glucagon-likepeptide-1(7-36)amide (truncated GLP-1; tGLP-1) are two importantinsulin-releasing hormones secreted from endocrine cells in theintestinal tract in response to feeding. Together with autonomic nervesthey play a vital supporting role to the pancreatic islets in thecontrol of blood glucose homeostasis and nutrient metabolism.

Dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) has been identified as akey enzyme responsible for inactivation of GIP and tGLP-1 in serum. DPPIV is completely inhibited in serum by the addition of diprotin A(DPA,0.1 mmol/l). This occurs through the rapid removal of the N-terminaldipeptides Tyr¹-Ala² and His⁷-Ala⁸ giving rise to the main metabolitesGIP(3-42) and GLP-1(9-36)amide, respectively. These truncated peptidesare reported to lack biological activity or to even serve as antagonistsat GIP or tGLP-1 receptors. The resulting biological half-lives of theseincretin hormones in vivo are therefore very short, estimated to be nolonger than 5 min.

In situations of normal glucose regulation and pancreatic B-cellsensitivity, this short duration of action is advantageous infacilitating momentary adjustments to homeostatic control. However, thecurrent goal of a possible therapeutic role of incretin hormones,particularly tGLP-1 in NIDDM therapy is frustrated by a number offactors in addition to finding a convenient route of administration.Most notable of these are rapid peptide degradation and rapid absorption(peak concentrations reached 20 min) and the resulting need for bothhigh dosage and precise timing with meals. Recent therapeutic strategieshave focused on precipitated preparations to delay peptide absorptionand inhibition of GLP-1 degradation using specific inhibitors of DPP IV.A possible therapeutic role is also suggested by the observation that aspecific inhibitor of DPP IV, isoleucine thiazolidide, lowered bloodglucose and enhanced insulin secretion in glucose-treated diabetic obeseZucker rats presumably by protecting against catabolism of the incretinhormones tGLP-1 and GIP.

Numerous studies have indicated that tGLP-1 infusion restores pancreaticB-cell sensitivity, insulin secretory oscillations and improved glycemiccontrol in various groups of patients with IGT or NIDDM. Longer termstudies also show significant benefits of tGLP-1 injections in NIDDM andpossibly IDDM therapy, providing a major incentive to develop an orallyeffective or long-acting tGLP-1 analogue. Several attempts have beenmade to produce structurally modified analogues of tGLP-1 which areresistant to DPP IV degradation. A significant extension of serumhalf-life is observed with His⁷-glucitol tGLP-1 and tGLP-1 analoguessubstituted at position 8 with Gly, Aib, Ser or Thr. However, thesestructural modifications seem to impair receptor binding andinsulinotrophic activity thereby compromising part of the benefits ofprotection from proteolytic degradation. In recent studies usingHis⁷-glucitol tGLP-1, resistance to DPP IV and serum degradation wasaccompanied by severe loss of insulin-releasing activity.

GIP shares not only the same degradation pathway as tGLP-1 but manysimilar physiological actions, including stimulation of insulin andsomatostatin secretion, and the enhancement of glucose disposal. Theseactions are viewed as key aspects in the antihyperglycemic properties oftGLP-1, and there is therefore good expectation that GIP may havesimilar potential as NIDDM therapy. Indeed, compensation by GIP is heldto explain the modest disturbances of glucose homeostasis observed intGLP-1 knockout mice. Apart from early studies, the anti-diabeticpotential of GIP has not been explored and tGLP-1 may seem moreattractive since it is viewed by some as a more potent insulinsecretagogue when infused at “so called” physiological concentrationsestimated by RIA.

The present invention aims to provide effective analogues of GIP. It isone aim of the invention to provide a pharmaceutical for treatment ofType 2 diabetes.

According to the present invention there is provided an effectivepeptide analogue of the biologically active GIP(1-42) which has improvedcharacteristics for treatment of Type 2 diabetes wherein the analoguecomprises at least 15 amino acid residues from the N terminus ofGIP(1-42) and has at least one amino acid substitution or modificationat position 1-3 and not including Tyr¹ glucitol GIP(1-42).

The structures of human and porcine GIP(1-42) are shown below. Theporcine peptide differs by just two amino acid substitutions atpositions 18 and 34.

The analogue may include modification by fatty acid addition at anepsilon amino group of at least one lysine residue.

The invention includes Tyr¹ glucitol GIP(1-42) having fatty acidaddition at an epsilon amino group of at least one lysine residue.

FIG 1. Primary structure of human gastric inhibitory polypeptide (GIP)     1               5                   10NH₂-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-            15                  20Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-     25Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-                        30                  35Trp-Lys-His-Asn-Ile-Thr-Gln-COOH                 40 FIG. 2. Primarystructure of porcine gastric inhibitory polypeptide (GIP)     1               5                  10NH2-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-            15                  20Ile-Ala-Met-Asp-Lys-Ile-Arg-Gln-Gln-Asp-Phe-Val-     25Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Ser-Asp-                        30                   35Trp-Lys-His-Asn-Ile-Thr-Gln-COOH                 40

Analogues of GIP(1-42) may have an enhanced capacity to stimulateinsulin secretion, enhance glucose disposal, delay glucose absorption ormay exhibit enhanced stability in plasma as compared to native GIP. Theyalso may have enhanced resistance to degradation.

Any of these properties will enhance the potency of the analogue as atherapeutic agent.

Analogues having D-amino acid substitutions in the 1, 2 and 3 positionsand/or N-glycated, N-alkylated, N-acetylated or N-acylated amino acidsin the 1 position are resistant to degradation in vivo.

Various amino acid substitutions at second and third amino terminalresidues are included, such as GIP(1-42)Gly2, GIP(1-42)Ser2,GIP(1-42)Abu2, GIP(1-42)Aib, GIP(1-42)D-Ala2, GIP(1-42)Sar2, andGIP(1-42)Pro3.

Amino-terminally modified GIP analogues include N-glycated GIP(1-42),N-alkylated GIP(1-42), N-actylated GIP(1-42), N-acetyl-GIP(1-42) andN-isopropyl GIP(1-42).

Other stabilised analogues include those with a peptide isostere bondbetween amino terminal residues at position 2 and 3. These analogues maybe resistant to the plasma enzyme dipeptidyl-peptidase IV (DPP IV) whichis largely responsible for inactivation of GIP by removal of theamino-terminal dipeptide Tyr1-Ala2.

In particular embodiments, the invention provides a peptide which ismore potent than human or porcine GIP in moderating blood glucoseexcursions, said peptide consisting of GIP(1-42) or N-terminal fragmentsof GIP(1-42) consisting of up to between 15 to 30 amino acid residuesfrom the N-terminus (i.e. GIP(1-15)-GIP(1-3)) with one or moremodifications selected from the group consisting of:

-   (a) substitution of Ala² by Gly-   (b) substitution of Ala² by Ser-   (c) substitution of Ala² by Abu-   (d) substitution of Ala² by Aib-   (e) substitution of Ala² by D-Ala-   (f) substitution of Ala² by Sar-   (g) substitution of Glu³ by Pro-   (h) modification of Tyr¹ by acetylation-   (i) modification of Tyr¹ by acylation-   (j) modification of Tyr¹ by alkylation-   (k) modification of Tyr¹ by glycation-   (l) conversion of Ala²-Glu³ bond to a psi [CH2NH] bond-   (m) conversion of Ala2-Glu3 bond to a stable peptide isotere bond-   (n) (n-isopropyl-H) 1GIP.

The invention also provides the use of Tyr¹-glucitol GIP in thepreparation of a medicament for the treatment of diabetes.

The invention further provides improved pharmaceutical compositionsincluding analogues of GIP with improved pharmacological properties.

Other possible analogues include certain commonly encountered aminoacids, which are not encoded by the genetic code, for example,beta-alanine (beta-ala), or other omega-amino acids, such as 3-aminopropionic, 4-amino butyric and so forth, ornithine (Orn), citrulline(Cit), homoarginine (Har), t-butylalanine (t-BuA), t-butylglycine(t-BuG), N-methylisoleucine (N-MeIle), phenylglycine (Phg), andcyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) andmethionine sulfoxide (MSO), substitution of the D form of a neutral oracidic amino acid or the D form of tyrosine for tyrosine.

According to the present invention there is also provided apharmaceutical composition useful in the treatment of diabetes type IIwhich comprises an effective amount of the peptide as described herein,in admixture with a pharmaceutically acceptable excipient.

The invention also provides a method of N-terminally modifying GIP oranalogues thereof the method comprising the steps of synthesizing thepeptide from the C terminal to the penultimate N terminal amino acid,adding tyrosine to a bubbler system as a F-moc protected Tyr(tBu)-Wangresin, deprotecting the N-terminus of the tyrosine and reacting with themodifying agent, allowing the reaction to proceed to completion,cleaving the modified tyrosine from the Wang resin and adding themodified tyrosine to the peptide synthesis reaction.

Preferably the agent is glucose, acetic anhydride or pyroglutamic acid.

The invention will now be demonstrated with reference to the followingnon-limiting example and the accompanying figures wherein:

FIG. 1 a illustrates degradation of GIP by DPP IV.

FIG. 1 b illustrates degradation of GIP and Tyr¹ glucitol GIP by DPP IV.

FIG. 2 a illustrates degradation of GIP human plasma.

FIG. 2 b illustrates degradation of GIP and Tyr¹-glucitol GIP by humanplasma.

FIG. 3 illustrates electrospray ionization mass spectrometry of GIP,Tyr¹-glucitol GIP and the major degradation fragment GIP(3-42).

FIG. 4 shows the effects of GIP and glycated GIP on plasma glucosehomeostasis.

FIG. 5 shows effects of GIP on plasma insulin responses.

FIG. 6 illustrates DPP-IV degradation of GIP 1-42.

FIG. 7 illustrates DPP-IV degradation of GIP (Abu²).

FIG. 8 illustrates DPP-IV degradation of GIP (Sar²).

FIG. 9 illustrates DPP-IV degradation of GIP (Ser²),

FIG. 10 illustrates DPP-IV degradation of N-Acetyl-GIP.

FIG. 11 illustrates DPP-IV degradation of glycated GIP.

FIG. 12 illustrates human plasma degradation of GIP.

FIG. 13 illustrates human plasma degradation of GIP (Abu²).

FIG. 14 illustrates human plasma degradation of GIP (Sar²).

FIG. 15 illustrates human plasma degradation of GIP (Ser²).

FIG. 16 illustrates human plasma degradation of glycated GIP.

FIG. 17 shows the effects of various concentrations of GIP 1-42 and GIP(Abu²) on insulin release from BRIN-BD11 cells incubated at 5.6 mMglucose.

FIG. 18 shows the effects of various concentrations of GIP 1-42 and GIP(Abu²) on insulin release from BRIN-BD11 cells incubated at 16.7 mMglucose.

FIG. 19 shows the effects of various concentrations of GIP 1-42 and GIP(Sar²) on insulin release from BRIN-BD11 cells incubated at 5.6 mMglucose.

FIG. 20 shows the effects of various concentrations of GIP 1-42 and GIP(Sar²) on insulin release from GRIN-BD11 cells incubated at 16.7 mMglucose.

FIG. 21 shows the effects of various concentrations of GIP 1-42 and GIP(Ser²) on insulin release from BRIN-BD11 cells incubated at 5.6 mMglucose.

FIG. 22 shows the effects of various concentrations of GIP 1-42 and GIP(Ser²) on insulin release from BRIN-BD11 cells incubated at 16.7 mMglucose.

FIG. 23 shows the effects of various concentrations of GIP 1-42 andN-Acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells incubated at5.6 mM glucose.

FIG. 24 shows the effects of various concentrations of GIP 1-42 andN-Acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells incubated at16.7 mM glucose.

FIG. 25 shows the effects of various concentrations of GIP 1-42 andglycated GIP 1-42 on insulin release from BRIN-BD11 cells incubated at5.6 mM glucose.

FIG. 26 shows the effects of various concentrations of GIP 1-42 andglycated GIP 1-42 on insulin release from GRIN-BD11 cells incubated at16.7 mM glucose.

FIG. 27 shows the effects of various concentrations of GIP 1-42 and GIP(Gly²) on insulin release from BRIN-BD11 cells incubated at 5.6 mMglucose.

FIG. 28 shows the effects of various concentrations of GIP 1-42 and GIP(Gly²) on insulin release from BRIN-BD11 cells incubated at 16.7 mMglucose.

FIG. 29 shows the effects of various concentrations of GIP 1-42 and GIP(Pro³) on insulin release from BRIN-BD11 cells incubated at 5.6 mMglucose.

FIG. 30 shows the effects of various concentrations of GIP 1-42 and GIP(Pro³) on insulin release from BRIN-BD11 cells incubated at 16.7 mMglucose.

EXAMPLE 1

Preparation of N-Terminally modified GIP and analogues thereof.

The N-terminal modification of GIP is essentially a three step process.Firstly, GIP is synthesised from its C-terminal (starting from aFmoc-Gln (Trt)-Wang resin, Novabiochem) up to the penultimate N-terminalamino-acid (Ala2) on an automated peptide synthesizer (AppliedBiosystems, CA, USA). The synthesis follows standard Fmoc peptidechemistry protocols. Secondly, the N-terminal amino acid of native GIP(Tyr) is added to a manual bubbler system as a Fmoc-protectedTyr(tBu)-Wang resin. This amino acid is deprotected at its N-terminus(piperidine in DMF (20% v/v)) and allowed to react with a highconcentration of glucose (glycation, under reducing conditions withsodium cyanoborohydride), acetic anhydride (acetylation), pyroglutamicacid (pyroglutamyl) etc. for up to 24 h as necessary to allow thereaction to go to completion. The completeness of reaction will bemonitored using the ninhydrin test which will determine the presence ofavailable free α-amino groups. Thirdly, (once the reaction is complete)the now structurally modified Tyr is cleaved from the wang resin (95%TFA, and 5% of the appropriate scavengers. N.B. Tyr is considered to bea problematic amino acid and may need special consideration) and therequired amount of N-terminally modified-Tyr consequently added directlyto the automated peptide synthesiser, which will carry on the synthesis,thereby stitching the N-terminally modified-Tyr to the α-amino ofGIP(Ala2), completing the synthesis of the GIP analogue. This peptide iscleaved off the Wang resin (as above) and then worked up using thestandard Buchner filtering, precipation, rotary evaporation and dryingtechniques.

EXAMPLE 2

The following example investigates preparation of Tyr¹-glycitol GIPtogether with evaluation of its antihyperglycemic and insulin-releasingproperties in vivo. The results clearly demonstrate that this novel GIPanalogue exhibits a substantial resistance to aminopeptidase degradationand increased glucose lowering activity compared with the native GIP.

Research Design and Methods

Materials. Human GIP was purchased from the American Peptide Company(Sunnyvale, Calif., USA). HPLC grade acetonitrile was obtained fromRathburn (Walkersburn, Scotland). Sequencing grade trifluoroacetic acid(TFA) was obtained from Aldrich (Poole, Dorset, UK). All other chemicalspurchased including dextran T-70, activated charcoal, sodiumcyanoborohydride and bovine serum albumin fraction V were from Sigma(Poole, Dorset, UK). Diprotin A (DPA) was purchased fromCalbiochem-Novabiochem (UK) Ltd. (Beeston, Nottingham, UK) and ratinsulin standard for RIA was obtained form Novo Industria (Copenhagen,Denmark). Reversed-phase Sep-Pak cartridges (C-18) were purchased fromMillipore-Waters (Milford, Mass., USA). All water used in theseexperiments was purified using a Milli-Q, Water Purification System(Millipore Corporation, Milford, Mass., USA).

Preparation of Tyr¹-glucitol GIP. Human GIP was incubated with glucoseunder reducing conditions in 10 mmol/l sodium phosphate buffer at pH 7.4for 24 h. The reaction was stopped by addition of 0.5 mol/l acetic acid(30 μl) and the mixture applied to a Vydac (C-18)(4.6×250 mm) analyticalHPLC column (The Separations Group, Hesperia, Calif., USA) and gradientelution conditions were established using aqueous/TFA andacetonitrile/TFA solvents. Fractions corresponding to the glycated peakswere pooled, taken to dryness under vacuum using an AES 1000 Speed-Vacconcentrator (Life Sciences International, Runcorn, UK) and purified tohomogeneity on a Supelcosil (C-8) (4.6×150 mm) column (Supelco Inc.,Poole, Dorset, UK).

Degradation of GIP and Tyr¹-glucitol GIP by DPP IV. HPLC-purified GIP orTyr¹-glucitol GIP were incubated at 37° C. with DPP-IV (5 mU) forvarious time periods in a reaction mixture made up to 500 μl with 50mmol/l triethanolamine-HCl, pH 7.8 (final peptide concentration 1mmol/l). Enzymatic reactions were terminated after 0, 2, 4 and 12 hoursby addition of 5 μl of 10% (v/v) TFA/water. Samples were made up to afinal volume of 1.0 ml with 0.12% (v/v) TFA and stored at −20° C. priorto HPLC analysis.

Degradation of GIP and Tyr¹-glucitol GIP by human plasma. Pooled humanplasma (20 μl) taken from six healthy fasted human subjects wasincubated at 37° C. with GIP or Tyr¹-glucitol GIP (10 μg) for 0 and 4hours in a reaction mixture made up to 500 μl, containing 50 mmol/ltriethanolamine/HCL buffer pH 7.8. Incubations for 4 hours were alsoperformed in the presence of diprotin A (5 mU). The reactions wereterminated by addition of 5 μl of TFA and the final volume adjusted to1.0 ml using 0.1% v/v TFA/water. Samples were centrifuged (13,000 g, 5min) and the supernatant applied to a C-18 Sep-Pak cartridge(Millipore-Waters) which was previously primed and washed with 0.1%(v/v) TFA/water. After washing with 20 ml 0.12% TFA/water, boundmaterial was released by elution with 2 ml of 80% (v/v)acetonitrile/water and concentrated using a Speed-Vac concentrator(Runcorn, UK). The volume was adjusted to 1.0 ml with 0.12% (v/v)TFA/water prior to HPLC purification.

HPLC analysis of degraded GIP and Tyr¹-glucitol GIP. Samples wereapplied to a Vydac C-18 widepore column equilibriated with 0.12% (v/v)TFA/H₂O at a flow rate of 1.0 ml/min. Using 0.1% (v/v) TFA in 70%acetonitrile/H₂O, the concentration of acetonitrile in the elutingsolvent was raised from 0% to 31.5% over 15 min, to 38.5% over 30 minand from 38.5% to 70% over 5 min, using linear gradients. The absorbancewas monitored at 206 nm and peak areas evaluated using a model 2221 LKBintegrator. Samples recovered manually were concentrated using aSpeed-Vac concentrator.

Electrospray ionization mass spectrometry (ESI-MS). Samples for ESI-MSanalysis containing intact and degradation fragments of GIP (from DPP IVand plasma incubations) as well as Tyr¹-glucitol GIP, were furtherpurified by HPLC. Peptides were dissolved (approximately 400 μmol) in100 μl of water and applied to the LCQ benchtop mass spectrometer(Finnigan MAT, Hemel Hempstead, UK) equipped with a microbore C-18 HPLCcolumn (150×2.0 mm, Phenomenex, UK, Ltd, Macclesfield). Samples (30 μldirect loop injection) were injected at a flow rate of 0.2 ml/min, underisocratic conditions 35% (v/v) acetonitile/water. Mass spectra wereobtained from the quadripole ion trap mass analyzer and recorded.Spectra were collected using full ion scan mode over the mass-to-charge(m/z) range 150-2000. The molecular masses of GIP and related structureswere determined from ESI-MS profiles using prominent multiple chargedions and the following equation M_(r)=iM_(i)−iM_(h) (whereM_(r)=molecular mass; M_(i)=m/z ratio; i=number of charges; M_(h)=massof a proton).

In vivo biological activity of GIP and Try¹-glucitol GIP. Effects of GIPand Tyr¹-glucitol GIP on plasma glucose and insulin concentrations wereexamined using 10-12 week old male Wistar rats. The animals were housedindividually in an air conditioned room and 22±2° C. with a 12 hourlight/12 hour dark cycle. Drinking water and a standard rodentmaintenance diet (Trouw Nutrition, Belfast) were supplied ad libitum.Food was withdrawn for an 18 hour period prior to intraperitonealinjection of glucose alone (18 mmol/kg body weight) or in combinationwith either GIP or Tyr¹-glucitol GIP (10 nmol/kg). Test solutions wereadministered in a final volume of 8 ml/kg body weight. Blood sampleswere collected at 0, 15, 30 and 60 minutes from the cut tip of the tailof conscious rats into chilled fluoride/heparin microcentrifuge tubes(Sarstedt, Nümbrecht, Germany). Samples were centrifuged using a Beckmanmicrocentrifuge for about 30 seconds at 13,000 g. Plasma samples werealiquoted and stored at −20° C. prior to glucose and insulindeterminations. All animal studies were done in accordance with theAnimals (Scientific Procedures) Act 1986.

Analyses. Plasma glucose was assayed by an automated glucose oxidaseprocedure using a Beckman Glucose Analyzer II [33]. Plasma insulin wasdetermined by dextran charcoal radioimmunoassay as described previously[34]. Incremental areas under plasma glucose and insulin curves (AUC)were calculated using a computer program (CAREA) employing thetrapezoidal rule [35] with baseline subtraction. Results are expressedas mean±SEM and values were compared using the Student's unpairedt-test. Groups of data were considered to be significantly different ifP<0.05.

Results

Degradation of GIP and Tyr¹-glucitol GIP by DPP IV. FIG. 1 illustratesthe typical peak profiles obtained from the HPLC separation of theproducts obtained from the incubation of GIP (FIG. 1 a) or Tyr¹-glucitolGIP (FIG. 1 b) with DPP IV for 0, 2, 4 and 12 hours. The retention timesof GIP and Tyr¹-glucitol GIP at t=0 were 21.93 minutes and 21.75 minutesrespectively. Degradation of GIP was evident after 4 hours incubation(54% intact), and by 12 hours the majority (60% of intact GIP wasconverted to the single product with a retention time of 21.61 minutes.Tyr¹-glucitol GIP remained almost completely intact throughout 2-12hours incubation. Separation was on a Vydac C-18 column using lineargradients of 0% to 31.5% acetonitrile over 15 minutes, to 38.5% over 30minutes and from 38.5 to 70% acetonitrile over 5 minutes.

Degradation of GIP and Tyr¹-glucitol GIP by human plasma. FIG. 2 shows aset of typical HPLC profiles of the products obtained from theincubation of GIP or Tyr¹-glucitol GIP with human plasma for 0 and 4 h.GIP (FIG. 2 a) with a retention time of 22.06 min was readilymetabolised by plasma within 4 hours incubation giving rise to theappearance of a major degradation peak with a retention time of 21.74minutes. In contrast, the incubation of Tyr¹-glucitol GIP under similarconditions (FIG. 2 b) did not result in the formation of any detectabledegradation fragments during this time with only a single peak beingobserved with a retention time of 21.77 minutes. Addition of diprotin A,a specific inhibitor of DPP IV, to GIP during the 4 hours incubationcompletely inhibited degradation of the peptide by plasma. Peakscorresponding with intact GIP, GIP (3-42) and Tyr¹-glucitol GIP areindicated. A major peak corresponding to the specific DPP IV inhibitortripeptide DPA appears in the bottom panels with retention time of 16-29min.

Identification of GIP degradation fragments by ESI-MS. FIG. 3 shows themonoisotopic molecular masses obtained for GIP, (panel A), Tyr¹-glucitolGIP (panel B) and the major plasma degradation fragment of GIP (panel C)using ESI-MS. The peptides analyzed were purified from plasmaincubations as shown in FIG. 2. Peptides were dissolved (approximately400 μmol) in 100 μl of water and applied to the LC/MS equipped with amicrobore C-18 HPLC column. Samples (30 μl direct loop injection) wereapplied at a flow rate of 0.2 ml/min, under isocratic conditions 35%acetonitrile/water. Mass spectra were recorded using a quadripole iontrap mass analyzer. Spectra were collected using full ion scan mode overthe mass-to-charge (m/z) range 150-2000. The molecular masses (M_(r)) ofGIP and related structures were determined from ESI-MS profiles usingprominent multiple charged ions and the following equationM_(r)=iM_(i)−iM_(h). The exact molecular mass (M_(r)) of the peptideswere calculated using the equation M_(r)=1M_(i)−iM_(h) as defined inResearch Design and Methods. After spectral averaging was performed,prominent multiple charges species (M+3H)³⁺ and (M+4H)⁴⁺ were detectedfrom GIP at m/z 1661.6 and 1246.8, corresponding to intact M_(r) 4981.8and 4983.2 Da, respectively (FIG. 3A). Similarly, for Tyr¹-glucitol GIP((M+4H)⁴⁺ and (M+5H)⁵⁺) were detected at m/z 1287.7 and 1030.3,corresponding to intact molecular masses of M^(r) 5146.8 and 5146.5 Da,respectively (FIG. 3B). The difference between the observed molecularmasses of the quadruply charged GIP and the N-terminally modified GIPspecies (163.6 Da) indicated that the latter peptide contained a singleglucitol adduct corresponding to Tyr¹-glucitol GIP. FIG. 3C shows theprominent multiply charged species (M+3H)³⁺ and (M+4H)⁴⁺ detected fromthe major fragment of GIP at m/z 1583.8 and 1188.1, corresponding tointact M^(r) 4748.4 and 4748 Da, respectively (FIG. 3C). Thiscorresponds with the theoretical mass of the N-terminally truncated formof the peptide GIP(3-42). This fragment was also the major degradationproduct of DPP IV incubations (data not shown).

Effects of GIP and Tyr¹-glucitol GIP on plasma glucose homeostasis.FIGS. 4 and 5 show the effects of intraperitoneal (ip) glucose alone (18mmol.kg) (control group), and glucose in combination with GIP orTyr¹-glucitol GIP (10 nmol/kg) on plasma glucose and insulinconcentrations.

(4A) Plasma glucose concentrations after i.p. glucose alone (18 mmol/kg)(control group), or glucose in combination with either GIP ofTyr¹-glucitol GIP (10 nmol/kg). The time of injection is indicated bythe arrow (0 min). (4B) Plasma glucose AUC calues for 0-60 min postinjection. Values are mean±SEM for six rats. **P<0.01, ***P<0.001compared with GIP and Tyr¹-glucitol GIP; tP<0.05, ttP<0.01 compared withnon-glucated GIP.

(5A). Plasma insulin concentrates after i.p. glucose along (18 mmol/kg)(control group), or glucose in combination with either with GIP orglycated GIP (10 nmol/kg). The time of injection is indicated by thearrow. (5B) Plasma insulin AUC values were calculated for each of the 3groups up to 90 minutes post injection. The time of injection isindicated by the arrow (0 min). Plasma insulin AUC values for 0-60 minpost injection. Values are mean±SEM for six rats. *P<0.05, **P<0.001compared with GIP and Tyr¹-glucitol GIP; tP<0.05, ttP<0.01 compared withnon-glycated GIP.

Compared with the control group, plasma glucose concentrations and areaunder the curve (AUC) were significantly lower following administrationof either GIP or Tyr¹-glucitol GIP (FIG. 4A, B). Furthermore, individualvalues at 15 and 30 minutes together with AUC were significantly lowerfollowing administration of Tyr¹-glucitol GIP as compared to GIP.Consistent with the established insulin-releasing properties of GIP,plasma insulin concentrations of both peptide-treated groups weresignificantly raised at 15 and 30 minutes compared with the values afteradministration of glucose alone (FIG. 5A). The overall insulinresponses, estimated as AUC were also signigicantly greater for the twopeptide-treated groups (FIG. 5B). Despite lower prevailing glucoseconcentrations than the GIP-treated group, plasma insulin response,calculated as AUC, following Tyr¹-glucitol GIP was significantly greaterthan after GIP (FIG. 5B). The significant elevation of plasma insulin at30 minutes is of particular note, suggesting that the insulin-releasingaction of Tyr¹-glucitol GIP is more protracted than GIP even in the faceof a diminished glycemic stimulus (FIGS. 4A, 5A).

Discussion

The forty two amino acid GIP is an important incretin hormone releasedinto the circulation from endocrine K-cells of the duodenum and jejunumfollowing ingestion of food. The high degree of structural conservationof GIP among species supports the view that this peptide plays andimportant role in metabolism. Secretion of GIP is stimulated directly byactively transported nutrients in the gut lumen without a notable inputfrom autonomic nerves. The most important stimulants of GIP release aresimple sugars and unsaturated long chain fatty acids, with amino acidsexerting weaker effects. As with tGLP-1, the insulin-releasing effect ofGIP is strictly glucose-dependent. This affords protection againsthypoglycemia and thereby fulfils one of the most desirable features ofany current or potentially new antidiabetic drug.

The present results demonstrate for the first time that Tyr¹-glucitolGIP displays profound resistance to serum and DPP IV degradation. UsingESI-MS the present study showed that native GIP was rapidly cleaved invitro to a major 4748.4 Da degradation product, corresponding toGIP(3-42) which confirmed previous findings using matrix-assisted laserdesorption ionization time-of-flight mass spectrometry. Serumdegradation was completely inhibited by diprotin A (Ile-Pro-Ile), aspecific competitive inhibitor of DPP IV, confirming this as the mainenzyme for GIP inactivation in vivo. In contrast, Tyr¹-glucitol GIPremained almost completely intact after incubation with serum or DPP IVfor up to 12 hours. This indicates that glycation of GIP at theamino-terminal Tyr¹ residue masks the potential cleavage site from DPPIV and prevents removal of the Tyr¹-Ala² dipeptide from the N-terminuspreventing the formation of GIP(3-42).

Consistent with in vitro protection against DPP IV, administration ofTyr¹-glucitol GIP significantly enhanced the antihyperglycemic activityand insulin-releasing action of the peptide when administered withglucose to rats. Native GIP enhanced insulin release and reduced theglycemic excursion as observed in many previous studies. However,amino-terminal glycation of GIP increased the insulin-releasing andantihyperglycemic actions of the peptide by 62% and 38% respectively, asestimated from AUC measurements. Detailed kinetic analysis is difficultdue to necessary limitation of sampling times, but the greater insulinconcentrations following Tyr¹-glucitol GIP as opposed to GIP at 30minutes post-injection is indicative of a longer half-life. The glycemicrise was modest in both peptide-treated groups and glucoseconcentrations following injection of Tyr¹-glucitol GIP wereconsistently lower than after GIP. Since the insulinotropic actions ofGIP are glucose-dependent, it is likely that the relativeinsulin-releasing potency of Tyr¹-glucitol GIP is greatly underestimatedin the present in vivo experiments.

In vitro studies in the laboratory of the present inventors usingglucose-responsive clonal B-cells showed that the insulin-releasingpotency of Tyr¹-glucitol GIP was several order of magnitude greater thanGIP and that its effectiveness was more sensitive to change of glucoseconcentrations within the physiological range. Together with the presentin vivo observations, this suggests that N-terminal glycation of GIPconfers resistance to DPP IV degradation whilst enhancing receptorbinding and insulin secretory effects on the B-cell. These attributes ofTyr¹-glucitol GIP are fully expressed in vivo where DPP IV resistanceimpedes degradation of the peptide to GIP(3-42), thereby prolonging thehalf-life and enhancing effective concentrations of the intactbiologically active peptide. It is thus possible that glycated GIP isenhancing insulin secretion in vivo both by enhanced potency at thereceptor as well as improving DPP IV resistance. Thus numerous studieshave shown that GIP (3-42) and other N-terminally modified fragments,including GIP(4-42), and GIP (17-42) are either weakly effective orinactive in stimulating insulin release. Furthermore, evidence existsthat N-terminal deletions of GIP result in receptor antagonistproperties in GIP receptor transfected Chinese hamster kidney cells [9],suggesting that inhibition of GIP catabolism would also reduce thepossible feedback antagonism at the receptor level by the truncatedGIP(3-42).

In addition to its insulinotopic actions, a number of other potentiallyimportant extrapancreatic actions of GIP may contribute to the enhancedantihyperglycemic activity and other beneficial metabolic effects ofTyr¹-glucitol GIP. These include the stimulation of glucose uptake inadipocytes, increased synthesis of fatty acids and activation oflipoprotein lipase in adipose tissue. GIP also promotes plasmatriglyceride clearance in response to oral fat loading. In liver, GIPhas been shown to enhance insulin-dependent inhibition ofglycogenolysis. GIP also reduces both glucagon-stimulated lipolysis inadipose tissue as well as hepatic glucose production. Finally, recentfindings indicate that GIP has a potent effect on glucose uptake andmetabolism in mouse isolated diaphragm muscle. This latter action may beshared with tGLP-1 and both peptides have additional benefits ofstimulating somatostatin secretion and slowing down gastric emptying andnutrient absorption.

In conclusion, this study has demonstrated for the first time that theglycation of GIP at the amino-terminal Tyr¹ residue limits GIPcatabolism through impairment of the proteolytic actions of serumpetidases and thus prolongs its half-life in vivo. This effect isaccompanied by enhanced antihyperglycemic activity and raised insulinconcentrations in vivo, suggesting that such DPP IV resistant analoguesshould be explored alongside tGLP-1 as potentially useful therapeuticagents for NIDDM. Tyr¹-glucitol GIP appears to be particularlyinteresting in this regard since such amino-terminal modification of GIPenhances rather than impairs glucose-dependent insulinotropic potency aswas observed recently for tGLP-1.

EXAMPLE 3

This example further looked at the ability of additional N-terminalstructural modifications of GIP in preventing inactivation by DPP and inplasma and their associated increase in both the insulin-releasingpotency and potential therapeutic value. Native human GIP, glycated GIP,acetylated GIP and a number of GIP analogues with N-terminal amino acidsubstitutions were tested.

Materials and Methods Reagents

High-performance liquid chromatography (HPLC) grade acetonitrile wasobtained from Rathburn (Walkersburn, Scotland). Sequencing gradetrifluoroacetic acid (TFA) was obtained from Aldrich (Poole, Dorset,UK). Dipeptidyl peptidase IV was purchased from Sigma (Poole, Dorset,UK), and Diprotin A was purchased from Calbiochem Novabiochem (Beeston,Nottingham, UK). RPMI 1640 tissue culture medium, foetal calf serum,penicillin and streptomycin were all purchased from Gibco (Paisley,Strathclyde, UK). All water used in these experiments was purified usinga Milli-Q, Water Purification System (Millipore, Millford, Mass., USA).

All other chemicals used were of the highest purity available.

Synthesis of GIP and N-Terminally Modified GIP Analogues

GIP, GIP(Abu2), GIP(Sar2), GIP(Ser2), GIP(Gly2) and GIP(Pro3) weresequentially synthesised on an Applied Biosystems automated peptidesynthesizer (model 432A) using standard solid-phase Fmoc procedure,starting with an Fmoc-Gln-Wang resin. Following cleavage from the resinby trifluoroacetic acid:water, thioanisole, ethanedithiol (90/2.5/5/2.5,a total volume of 20 ml/g resin), the resin was removed by filtrationand the filtrate volume was decreased under reduced pressure. Drydiethyl ether was slowly added until a precipitate was observed. Theprecipitate was collected by low-speed centrifugation, resuspended indiethyl ether and centrifuged again, this procedure being carried out atleast five times. The pellets were then dried in vacuo and judged pureby reversed-phase HPLC on a Waters Millennium 2010 chromatography system(Software version 2.1.5.). N-terminal glycated and acetylated GIP wereprepared by minor modification of a published method.

Electrospray ionization-mass spectrometry (ESI-MS) was carried out asdescribed in Example 2.

Degradation of GIP and novel GIP analogues by DPP IV and human plasmawas carried out as described in Example 2.

Culture of Insulin Secreting Cells

BRIN-BD11 cells [30] were cultured in sterile tissue culture flasks(Corning, Glass Works, UK) using RPMI-1640 tissue culture mediumcontaining 10% (v/v) foetal calf serum, 1% (v/v) antibiotics (100 U/mlpenicillin, 0.1 mg/ml streptomycin) and 11.1 mM glucose. The cells weremaintained at 37° C. in an atmosphere of 5% CO₂ and 95% air using a LEECincubator (Laboratory Technical Engineering, Nottingham, UK).

Acute Tests for Insulin Secretion

Before experimentation, the cells were harvested from the surface of thetissue culture flasks with the aid of trypsin/EDTA (Gibco), seeded into24-multiwell plates (Nunc, Roskilde, Denmark) at a density of 1.5×105cells per well, and allowed to attach overnight at 37° C. Acute testsfor insulin release were preceded by 40 min pre-incubation at 37° C. in1.0 ml Krebs Ringer bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mMCaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 10 mM NaHCO₃, 5 g/l bovine serumalbumin, pH 7.4) supplemented with 1.1 mM glucose. Test incubations wereperformed (n=12) at two glucose concentrations (5.6 mM and 16.7 mM) witha range of concentrations (10⁻¹³ to 10⁻⁸ M) of GIP or GIP analogues.After 20 min incubation, the buffer was removed from each well andaliquots (200 μl) were used for measurement of insulin byradioimmunoassay [31].

Statistical Analysis

Results are expressed as mean±S.E.M. and values were compared using theStudent's unpaired t-test. Groups of data were considered to besignificantly different if P<0.05.

Results and Discussion

Structural identification of GIP and GIP analogues by ESI-MS

The monoisotopic molecular masses of the peptides were determined usingESI-MS. After spectral averaging was performed, prominent multiplecharged species (M+3H)3+ and (M+4H)4+ were detected for each peptide.Calculated molecular masses confirmed the structural identity ofsynthetic GIP and each of the N-terminal analogues.

Degradation of GIP and Novel GIP Analogues by DPP-IV

FIGS. 6-11 illustrate the typical peak profiles obtained from the HPLCseparation of the reaction products obtained from the incubation of GIP,GIP(Abu2), GIP(Sar2), GIP(Ser2), glycated GIP and acetylated GIP withDPP IV, for 0, 2, 4, 8 and 24 h. The results summarised in Table 1indicate that glycated GIP, acetylated GIP, GIP(Ser2) are GIP(Abu2) moreresistant than native GIP to in vitro degradation with DPP IV. Fromthese data GIP(Sar2) appears to be less resistant.

Degradation of GIP and GIP Analogues by Human Plasma

FIGS. 12-16 show a representative set of HPLC profiles obtained from theincubation of GIP and GIP analogues with human plasma for 0, 2, 4, 8 and24 h. Observations were also made after incubation for 24 h in thepresence of DPA. These results are summarised in Table 2 are broadlycomparable with DPP IV incubations, but conditions which more closelymirror in vivo conditions are less enzymatically severe. GIP was rapidlydegraded by plasma. In comparison, all analogues tested exhibitedresistance to plasma degradation, including GIP(Sar2) which from DPP IVdata appeared least resistant of the peptides tested. DPA substantiallyinhibited degradation of GIP and all analogues tested with completeabolition of degradation in the cases of GIP(Abu2), GIP(Ser2) andglycated GIP. This indicates that DPP IV is a key factor in the in vivodegradation of GIP.

Dose-Dependent Effects of GIP and Novel GIP Analogues on InsulinSecretion

FIGS. 17-30 show the effects of a range of concentrations of GIP,GIP(Abu2), GIP(Sar2), GIP(Ser2), acetylated GIP, glycated GIP, GIP(Gly2)and GIP(Pro3) on insulin secretion from GRIN-BD11 cells at 5.6 and 16.7mM glucose. Native GIP provoked a prominent and dose-related stimulationof insulin secretion. Consistent with previous studies [28], theglycated GIP analogue exhibited a considerably greater insulinotropicresponse compared with native peptide. N-terminal acetylated GIPexhibited a similar pattern and the GIP(Ser2) analogue also evoked astrong response. From these tests, GIP(Gly2) and GIP(Pro3) appeared tothe least potent analogues in terms of insulin release. Other stableanalogues tested, namely GIP(Abu2) and GIP(Sar2), exhibited a complexpattern of responsiveness dependent on glucose concentration and doseemployed. Thus very low concentrations were extremely potent underhyperglycaemic conditions (16.7 mM glucose). This suggests that eventhese analogues may prove therapeutically useful in the treatment oftype 2 diabetes where insulinotropic capacity combined with in vivodegradation dictates peptide potency.

TABLE 1 % Intact peptide remaining after incubation with DPPIV % Intactpeptide remaining after time (h) Peptide 0 2 4 8 24 GIP 1-42 100 52 ± 123 ± 1 0 0 Glycated GIP 100 100 100 100 100 GIP (Abu²) 100 38 ± 1 28 ± 20 0 GIP (Ser²) 100 77 ± 2 60 ± 1 32 ± 4 0 GIP (Sar²) 100 28 ± 2  8 0 0N-Acetyl-GIP 100 100 100 100 0

TABLE 2 % Intact peptide remaining after incubation with human plasma %Intact peptide remaining after incubations with human plasma Peptide 0 24 8 24 DPA GIP 1-42 100 52 ± 1 23 ± 1 0 0 68 ± 2 Glycated GIP 100 100100 100 100 100 GIP (Abu²) 100 38 ± 1 28 ± 2 0 0 100 GIP (Ser²) 100 77 ±2 60 ± 1 32 ± 4 0 63 ± 3 GIP (Sar²) 100 28 ± 2  8 0 0 100

Tables represent the percentage of intact peptide (i.e. GIP 1-42)relative to the major degradation product GIP 3-42. Values were takenfrom HPLC traces performed in triplicate and the mean and S.E.M. valuescalculated. DPA is diprotin A, a specific inhibitor of DPPIV.

1-11. (canceled)
 12. A peptide analogue of GIP (1-42) comprising atleast 15 amino acid residues from the N-terminal end of GIP (1-42),wherein those amino acids present at positions 15-30 of the peptideanalogue are unsubstituted with respect to GIP (1-42), with the provisothat the peptide analogue is not tyrosine¹ glucitol GIP (1-42), andwherein (a) the peptide analogue comprises two amino acid substitutionsor modifications selected from the group consisting of: (i) an L- orD-amino acid substitution at position 1 selected from Alanine, Arginine,Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine,Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine,Threonine, Tryptophan, Tyrosine, Valine or an amino acid modification atposition 1; and one of an L- or D-amino acid substitution at position 2or 3 selected from Alanine, Arginine, Asparagine, Aspartic acid,Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine,Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine,Tryptophan, Tyrosine, Valine; or an amino acid modification at position2 or 3; and (ii) an amino acid substitution at position 2 selected fromAlanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid,Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine,Phenylalanine, Proline, Threonine, Tryptophan, Tyrosine, Valine or anamino acid modification at position 2; and one of an L- or D-amino acidmodification at position 1, an amino acid modification at position 1, anL- or D-amino acid modification at position 3, or an amino acidsubstitution at position 3; or wherein (b) the peptide analoguecomprises one amino acid substitution or modification selected from thegroup consisting of: an L- or D-amino acid substitution at position 1;an amino acid modification at position 1; an amino acid modification atposition 2; an L-amino acid substitution at position 2; an D-amino acidsubstitution at position 2 by a D-amino acid selected from D-arginine,D-asparagine, D-aspartic acid, D-cysteine, D-glutamic acid, D-glutamine,D-glycine, D-histidine, D-isoleucine, D-leucine, D-lysine, D-methionine,D-phenylalanine, D-proline, D-serine, D-threonine, D-tryptophan,D-tyrosine and D-valine; an L- or D-amino acid substitution at position3, and an amino acid modification at position 3; and wherein the peptideanalogue is DPP-IV resistant and is capable of binding a receptor ofGIP.
 13. The peptide analogue of claim 12, wherein the peptide analoguecomprises a peptide analogue consisting of up to between 15 to 30 aminoacids of GIP(1-42).
 14. The peptide analogue of claim 12, wherein thepeptide analogue activates the receptor of GIP to stimulate insulinrelease.
 15. The peptide analogue of claim 12, wherein the peptideanalogue is capable of binding a receptor of GIP and wherein the peptideanalogue comprises at least one amino acid substitution or modificationat one of position 1, 2 or 3, with the proviso that the peptide analogueis not tyrosine¹ glucitol GIP (1-42), wherein the amino acidsubstitution or modification is selected from the group consisting of:substitution at position 1 by an amino acid; substitution at position 2by an L-amino acid, amino isobutyric acid or sarcosine; substitution atposition 3 by an amino acid, amino isobutyric acid or sarcosine;conversion of the Ala²-Glu³ bond to a ψ[CH₂NH] bond; conversion of theAla²-Glu³ bond to a stable isostere bond; and substitution bybeta-alanine, an omega-amino acid, 3-amino propionic acid, 4-aminobutyric acid, ornithine, citrulline, homoarginine, t-butylalanine,t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine,norleucine, cysteic acid and methionine sulfoxide.
 16. The peptideanalogue of claim 12, with the proviso that, when the analogue comprisesone amino acid modification at position 1, the one modification is notglycation of the tyrosine residue at position
 1. 17. A method ofstimulating insulin release, the method comprising administering to anindividual an effective amount of the peptide analogue of claim
 12. 18.The method of claim 17, wherein the peptide analogue comprises a peptideanalogue consisting of up to between 15 to 30 amino acids of GIP(1-42).19. A method of moderating blood glucose excursions, the methodcomprising administering to an individual an effective amount of thepeptide analogue of claim
 12. 20. The method of claim 19, wherein thepeptide analogue comprises a peptide analogue consisting of up tobetween 15 to 30 amino acids of GIP(1-42).
 21. A method of treatingdiabetes comprising administering to an individual an effective amountof the peptide analogue of claim
 12. 22. The method of claim 21, whereinthe peptide analogue comprises a peptide analogue consisting of up tobetween 15 to 30 amino acids of GIP(1-42).
 23. The method of claim 21,wherein the diabetes is type 2 diabetes.
 24. The method of claim 22,wherein the diabetes is type 2 diabetes.